Paratuberculosis (Johne's disease) is an incurable, fatal disease of domestic and wild ruminants. Mycobacterium paratuberculosis (M. paratuberculosis) is the etiologic agent of this disease. Mycobacterium avium (M. avium) and M. paratuberculosis are slow-growing faculative intracellular mycobacteria able to grow in mononuclear phagocytes. DNA-DNA hybridization studies have shown that these micro-organisms belong to a single genomic species (Hurley et al., Intl. J. Syst. Bacteriol., 38:143-146, 1988), and it has been proposed to reclassify M. paratuberculosis as a subspecies of M. avium (Thorel et al., Intl. J. Syst. Bacteriol. 40:254-260, 1990). Furthermore, all M. paratuberculosis strains are characterized by the presence of the insertion sequence IS900 (Green et al., Nucleic Acids Res., 17:9063-9073, 1989), which is absent from most M. avium strains. Phenotypic differences between M. avium and M. paratuberculosis, such as mycobactin requirement, ability to grow on egg medium, growth stimulation by pyruvate, and tolerance to cycloserine correlate with variations in pathogenicity and host range (Thorel et al., Intl. J. Syst. Bacteriol. 40:254-260, 1990).
Johne's disease is manifested by chronic diarrhea and weight loss. After months of diarrhea and wasting, the affected animals either die or are culled. In the United States, the prevalence of M. paratuberculosis infection in dairy and beef cattle herds has reached 34% in certain areas (Collins et al., J. Am. Vet. Med. Assn. 187:323-329, 1992; Collins et al., J. Am. Vet. Med. Assn. 204:636-641, 1994) and results in millions of dollars in lost revenues annually. Furthermore, M. paratuberculosis has been tentatively linked to Crohn's disease, a chronic granulomatous ileitis in humans. Evidence supporting the possibility that M. paratuberculosis is the etiologic agent of Crohn's disease includes culture of M. paratuberculosis is from intestinal tissue, and amplification by PCR of the subspecies-specific IS900 sequence of M. paratuberculosis from biopsy specimens.
Natural infection in cattle is usually acquired in the first months of life. The age of onset of clinical Johne's disease varies, being most frequent during or after the second lactation in dairy cattle. The prolonged incubation time and the difficulty in diagnosing subclinical cases facilitate the insidious spread of the infection within a herd. Bacteriologic culture is the most definitive diagnostic method, but requires substantial time and labor (Stabel, J. Vet. Diag. Invest., 9:375-380, 1997), and it is unable to detect infected animals that do not shed acid-fast bacilli. Progress has been made by combining fecal culture, PCR detection (IS900), and tests for humoral (ELISA) or cellular immunity (IFN-γ test) (Collins, Proceedings of the Fifth Intl. Colloq. Paratuberculosis, Chiodini et al., eds., Intl. Assn. for Paratuberculosis, 1997, pp. 232-241.). More recently, a gene unique to M. paratuberculosis (hspX) was identified and has promise as a new diagnostic tool (Ellingson et al., Mol. Cell Probes 12:133-142, 1998).
Currently, treatment of paratuberculosis in cattle is limited to the extra label use of therapeutic agents (St.-Jean et al., Vet. Clin. N. Am. Food Anim. Pract., 7:793-804, 1991; St.-Jean, Vet. Clin. N. Am. Food Anim. Pract., 12:417-430, 1996), and no antibiotic treatment is recommended for clinical cases of Crohn's disease. Even with a prolonged drug regimen paratuberculosis in cattle is invariably fatal.
Little is known about M. paratuberculosis immunogens and virulence determinants (Cocito et al., Clin. Microbiol. Rev. 7:328-345, 1994). Lipoarabinoman (Sugden et al., J. Clin. Microbiol., 29:1659-1664, 1991), glycopeptidolipid 1 (Camphausen et al., Proc. Natl. Acad. Sci. USA, 82:3068-3072, 1985), and 35 kDa (p35) antigen (El Zaatari et al., J. Clin. Microbiol., 35:1794-1799, 1997) are three major immunogens. Antigen p35 was recognized by sera from all clinically diseased cattle and by fifteen out of twenty cattle with subclinical diseases. This antigen, however, is not specific for paratuberculosis, since it is widely present in other strains of the M. avium complex. Several protein antigens have been identified by two-dimensional immunoelectrophoresis with hyperimmune sera, but only a subset of these antigens are recognized by sera from animals with paratuberculosis (Gunnarsson and Fedstand, Acta Vet. Scand. 20:200-215, 1979). Comparison of the two-dimensional gel electrophoretic profiles of M. paratuberculosis and M. avium cells grown in Middlebrook 7H9 medium followed by Western blot analysis, using antiserum from clinically infected cows, revealed a 42 kDa protein which may be specific for M. paratuberculosis (White et al., Am. J. Vet. Res., 55:1399-1405, 1994). Using the same methodology, AHPC gene products were identified that may be antigenic (Hsieh et al., Proceed. Fifth Intl. Colloq. Paratuberculosis, Chiodini et al., eds., 1997, pp. 82-87).
M. paratuberculosis antigens that have been cloned include the heat shock proteins HSP65 and HSP70, the transposase form IS900, a putative serine protease, bacterioferritin, the 34 kDa antigen bearing major B cell epitopes (reviewed by Stevenson and Sharp, Vet. J. 153:269-286, 1997), and more recently, the 35 kDa antigen (El Zaatari et al., J. Clin. Microbiol. 35:1794-1799, 1997) and the fled protein from IS900 (Doran et al., Microbiol. 143:547-552, 1997). In addition, a novel extracellular ferric reductase enzyme activity with a potential role in the evasion of intracellular defense mechanisms has been identified (Homuth et al. Infect. Immun., 66:710-716, 1998). Secreted proteins of M. paratuberculosis have received attention as potential immune targets early in infection. Some of these proteins are present as glyconjugates and different epitopes in the glycosylated and non-glycosylated moieties seem to be recognized in cattle and sheep (Mutharia et al, Infect. Immun. 65:387-394, 1997).
Diagnosis and control of paratuberculosis presents a significant challenge. Although vaccination does reduce clinical signs of Johne's disease, it does not prevent losses in milk production (van Schaik et al., Vet. Rec., 139:624-627, 1996). Improved vaccines and diagnostic tools are urgently needed. Likewise faster, specific, and more accurate and sensitive diagnostics need to be developed, especially to detect animals in the early stages of the disease. These tools preferably should also be able to discriminate between vaccinated and infected animals.
Identification of M. paratuberculosis virulence determinants is a critical step in developing suitable methods of diagnosis and control, and requires a systematic method by which virulence determinants can be found. U.S. Pat. No. 5,783,386 to Jacobs et al. describes a method for identifying virulence determinants of mycobacterial species involving the preparation of a genomic DNA library and constructing shuttle vectors containing inserts from the library constructed. These vectors are then used to transform a virulent organisms to form recombinants. Virulence determinants are identified by inoculating animals with the transformed recombinant organisms to select virulent recombinants and then identifying the DNA sequences that confer virulence. Cavaignac, et al. (Arch. Microbial. 173:229-231, 2000) studied virulence mechanisms in M. paratuberculosis by the introduction of random mutants using transposon mutagenesis. Two thousand mutants were screened on the basis of auxotrophy and altered cell wall. Pelicic et al. (U.S. Pat. No. 6,096,549) disclose a method for inserting a transposon into a mycobacterium strain using a vector containing the sacB gene. Use of this vector to identify virulence determinants is also disclosed. The present disclosure teaches an alternative method utilizing transposon mediated mutation and positive selection of mutants by antimicrobial agents that kill growing mycobacteria.